PROJECT SUMMARY Congenital heart disease remains the most common birth defect and the major cause of birth defect-related deaths in the world, and originates at least in part from genetic defects altering the development and maturation of the heart. Despite substantial progress, there are still huge knowledge gaps in basic understanding of mechanisms controlling this process. While the roles of key cardiac-specific transcription factors in heart development have been carefully studied, the significance of protein modifiers in this process has not been recognized. The overall goal of this project is to uncover the essential role of NEDD8, a novel ubiquitin-like protein modifier, in heart development and maturation. NEDD8 modifies protein targets in a way similar to ubiquitination, termed neddylation. Neddylation requires NEDD8 specific E1, E2, and E3 enzymes. Through regulating the function of protein targets, neddylation participates in diverse cellular processes and pathophysiological events. However, whether neddylation plays any role in cardiac development is completely unknown. By targeting a subunit of the only NEDD8 E1 activating enzyme (NAE1), our pilot studies uncover a critical role of neddylation in maintaining the structural integrity and function of neonatal hearts. The importance of neddylation in the heart is connected to its novel function in regulation of Hippo-YAP signaling, a conserved and crucial pathway that controls heart development. Therefore, this project is to test the central hypothesis that NAE1 regulates embryonic cardiac development through sustaining CM proliferation in a YAP-dependent manner. Two specific aims are proposed to test this hypothesis. Aim 1 will establish the functional importance of neddylation in embryonic heart development. Using a newly created NAE1 knockout mouse line, we will thoroughly characterize the impact of NAE1 deficiency on a series of concordant and sequential cardiac morphogenic events. The cardiac phenotype will be linked to defects, if any, in CM differentiation and proliferation. Moreover, we will identify novel pathways under the regulation of neddylation in the developing heart. In Aim 2, we will delineate molecular mechanisms by which neddylation controls CM proliferation, with a focus on the Hippo-YAP pathway. We will determine the effect of NAE1 deficiency on Hippo-YAP signaling in vivo and dissect molecular mechanisms of how neddylation controls this pathway. We will also test whether reactivation of YAP signaling rescues NAE1 inactivation-induced CM proliferation arrest. Given the extensive preliminary data gathered, the strong and experienced mentoring team, as well as the high caliber of the training plan, this proposal is likely to prove successful. Completion of this study will provide the applicant comprehensive training in various aspects of cardiovascular biology, which forms the basis of setting his long-term career goal of becoming an independent physician scientist. The proposed study will, for the first time, establish the essential role of neddylation in cardiac development, offering novel mechanisms underlying the development of CHD, one of the most important causes of birth-related morbidity and mortality worldwide.